Gating of Cl^- Currents in Protoplasts from the Marine Alga Valonia utricularis Depends on the Transmembrane Cl^- Gradient and Is Affected by Enzymatic Cell Wall Degradation

The electrical properties of protoplasts of the turgor pressure-regulating giant marine alga Valonia utricularis were investigated by using the patch-clamp technique. In the whole-cell configuration, large inward currents were elicited by negative-going voltage pulses. The time-dependent component was predominantly carried by Cl^-, as revealed by 'tail current' analysis. When experiments were performed on protoplasts directly after mechanical release from the 'mother cell', small outward currents were additionally observed at membrane voltages more positive than ECl^-. These outward currents disappeared to a large extent after treatment of the protoplasts with a mixture of cell wall-degrading enzymes. Plots of the chord conductance versus the clamped membrane voltage revealed that enzymatic treatment affected the gating properties. By fitting Boltzmann distributions to the data, a midpoint potential of + 5 ± 5 mV (n = 7) was obtained in symmetrical Cl^- solutions for mechanically released protoplasts. In contrast, protoplasts treated additionally with enzymes exhibited a midpoint potential of -13 ± 5 mV (n = 8). By varying the external and internal Cl^- concentration, gating was also shown to depend on the Cl^- gradient across the plasmalemma both in enzymatically treated and untreated protoplasts. Plotting of the midpoint potential against the Nernst potential of Cl^- rendered a slope less than 1 (0.70 and 0.64, respectively) indicating that gating did not strictly depend on the electrochemical Cl^- gradient. The voltage- and Cl^--dependence as well as inhibition experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) suggested that the Cl^- conductance of the membrane is dominated by the Valonia Anion Channel 1 (VAC1) described by Heidecker, M., Wegner, L.H., Zimmermann, U. 1999: A patch-clamp study of ion channels in proto-plasts prepared from the marine alga Valonia utricularis. J. Membrane Biol. 172:235–247. The relevance of the findings for membrane potential control and turgor regulation in V. utricularis as well as the general implications of the data for electrophysiological work on protoplasts (that are usually obtained by enzymatic digestion of plant tissue) are discussed.